The present invention relates generally to a novel phosphodiesterase (PDE) designated PDE10. Depending on nomenclature used, PDE10 is also referred to as PDE9.
Phosphodiesterases (PDEs) hydrolyze 3xe2x80x2, 5xe2x80x2 cyclic nucleotides to their respective nucleoside 5xe2x80x2 monophosphates. The cyclic nucleotides cAMP and cGMP are synthesized by adenylyl and guanylyl cyclases, respectively, and serve as second messengers in a number of cellular signaling pathways. The duration and strength of the second messenger signal is a function of the rate of synthesis and the rate of hydrolysis of the cyclic nucleotide.
Multiple families of PDEs have been identified. The nomenclature system includes first a number that indicates the PDE family. To date, nine families (PDE1-9) are known which are classified by: (i) primary structure; (ii) substrate preference; (iii) response to different modulators; (iv) sensitivity to specific inhibitors; and (v) modes of regulation [Loughney and Ferguson, in Phosphodiesterase Inhibitors, Schudt, et al. (Eds.), Academic Press: New York, N.Y. (1996) pp.1-19]. The number indicating the family is followed by a capital letter, indicating a distinct gene, and the capital letter followed by a second number, indicating a specific splice variant or a specific transcript that utilizes a unique transcription initiation site.
The amino acid sequences of all mammalian PDEs identified to date include a highly conserved region of approximately 270 amino acids located in the carboxy terminal half of the protein [Charbonneau, et al., Proc. Natl. Acad. Sci. (USA) 83:9308-9312 (1986)]. The conserved domain includes the catalytic site for cAMP and/or cGMP hydrolysis and two putative zinc binding sites as well as family specific determinants [Beavo, Physiol. Rev. 75:725-748 (1995); Francis, et al., J. Biol. Chem. 269:22477-22480 (1994)]. The amino terminal regions of the various PDEs are highly variable and include other family specific determinants such as: (i) calmodulin binding sites (PDE1); (ii) non-catalytic cyclic GMP binding sites (PDE2, PDE5, PDE6); (iii) membrane targeting sites (PDE4); (iv) hydrophobic membrane association sites (PDE3); and (v) phosphorylation sites for either the calmodulin-dependent kinase II (PDE1), the cAMP-dependent kinase (PDE1, PDE3, PDE4), or the cGMP dependent kinase (PDE5) [Beavo, Physiol. Rev. 75:725-748 (1995); Manganiello, et al., Arch. Biochem. Acta 322:1-13 (1995); Conti, et al., Physiol. Rev. 75:723-748 (1995)].
Members of the PDE1 family are activated by calcium-calmodulin. Three genes have been identified; PDE1A and PDE1B preferentially hydrolyze cGMP while PDE1C has been shown to exhibit a high affinity for both cAMP and cGMP. The PDE2 family is characterized as being specifically stimulated by cGMP [Loughney and Ferguson, supra]. Only one gene has been identified, PDE2A, the enzyme product of which is specifically inhibited by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Enzymes in the PDE3 family are specifically inhibited by cGMP. Two genes are known, PDE3A and PDE3B, both having high affinity for both cAMP and cGMP, although the Vmax for cGMP hydrolysis is low enough that cGMP functions as a competitive inhibitor for cAMP hydrolysis. PDE3 enzymes are specifically inhibited by milrinone and enoximone [Loughney and Ferguson, supra]. The PDE4 family effects cAMP hydrolysis and includes four genes, PDE4A, PDE4B, PDE4C, and PDE4D, each having multiple splice variants. Members of this family are specifically inhibited by the anti-depressant drug rolipram. Members of PDE5 family bind cGMP at non-catalytic sites and preferentially hydrolyze cGMP. Only one gene, PDE5A, has been identified. The photoreceptor PDE6 enzymes specifically hydrolyze cGMP [Loughney and Ferguson, supra]. Genes include PDE6A and PDE6B (the protein products of which dimerize and bind two copies of a smaller xcex3 inhibitory subunit to form rod PDE), in addition to PDE6C which associates with three smaller proteins to form cone PDE. The PDE7 family effects cAMP hydrolysis but, in contrast to the PDE4 family, is not inhibited by rolipram [Loughney and Ferguson, supra]. Only one gene, PDE7A, has been identified. The PDE8 family has been shown to hydrolyze both cAMP and cGMP and is insensitive to inhibitors specific for PDEs 1-5. Depending on nomenclature used, PDE8 is also referred to as PDE10, but is distinct from PDE10 described herein. The PDE9 family preferentially hydrolyzes cAMP and is not sensitive to inhibition by rolipram, a PDE4-specific inhibitor, or isobutyl methyl xanthine (IBMX), a non-specific PDE inhibitor. Depending on nomenclature used, PDE9 is also referred to as PDE8, but is distinct from PDE8 mentioned above. To date, two genes have been identified in the PDE9 family.
Specific and non-specific inhibitors of the various PDE protein families have been shown to be effective in treating disorders attributable, in part, to aberrant levels of cAMP or cGMP. For example, the PDE4-specific inhibitor rolipram, mentioned above as an anti-depressant, inhibits lipopolysaccharide-induced expression of TNFxcex1 and has been effective in treating multiple sclerosis in an animal model. Other PDE4-specific inhibitors are being investigated for use as anti-inflammatory therapeutics, and efficacy in attenuating the late asthmatic response to allergen challenge has been demonstrated [Harbinson, et al., Eur. Respir. J. 10:1008-1014 (1997)]. Inhibitors specific for the PDE3 family have been approved for treatment of congestive heart failure. PDE5 inhibitors are currently being evaluated for treatment of penile erectile dysfunction [Boolell, et al., Int. J. Impotence Res. 8:47-50 (1996)]. Non-specific inhibitors, such as theophylline and pentoxifylline, are currently used in the treatment of respiratory and vascular disorders, respectively.
Given the importance of cAMP and cGMP in intracellular second messenger signaling, there thus exists an ongoing need in the art to identify additional PDE species. Identification of heretofore unknown families of PDEs, and genes and splice variants thereof, will provide additional pharmacological approaches to treating conditions in which cyclic nucleotide pathways are aberrant, as well as conditions in which modulation of intracellular cAMP and/or cGMP levels in certain cell types is desirable. Identification of family-specific and enzyme-specific inhibitors will permit development of therapeutic and prophylactic agents which act on desired cell types expressing PDEs and/or particular metabolic pathways regulated by cyclic nucleotide monophosphate steady-state concentrations.
In brief, the prevent invention provides purified and isolated PDE10 polypeptides. Preferred polypeptides comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23.
The invention also provides polynucleotides encoding polypeptides of the invention. A preferred polynucleotide comprises the sequence set forth in SEQ ID NO: 1. Polynucleotides of the invention include polynucleotides encoding a human PDE10 polypeptide selected from the group consisting of: a) the polynucleotide according to SEQ ID NO: 1, 18, 20 or 22; b) a DNA which hybridizes under moderately stringent conditions to the non-coding strand of the polynucleotide of (a); and c) a DNA which would hybridize to the non-coding strand of the polynucleotide of (a) but for the redundancy of the genetic code. Polynucleotides of the invention comprise any one of the polynucleotide set out in SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 22, as well as fragments thereof. The invention provide polynucleotides which are DNA molecules. DNA molecules include cDNA, genomic DNA, and wholly or partially chemically synthesized DNA molecule. The invention also provides antisense polynucleotides which specifically hybridizes with the complement of a polynucleotide of the invention.
The invention also provides expression constructs comprising a polynucleotide of the invention, host cells transformed or transfected with an expression construct of the invention, and methods for producing a PDE10 polypeptide comprising the steps of: a) growing the host cell of the invention under conditions appropriate for expression of the PDE10 polypeptide and b) isolating the PDE10 polypeptide from the host cell or the medium of its growth.
The invention further provides antibodies specifically immunoreactive with a polypeptide of the invention. Preferably, the antibody is a monoclonal antibody. The invention also provides hybridomas which produces an antibody of the invention. Anti-idiotype antibody specifically immunoreactive with the antibody of the invention are also contemplated.
The invention also provides methods to identify a specific binding partner compound of a PDE10 polypeptide comprising the steps of: a) contacting the PDE10 polypeptide with a compound under conditions which permit binding between the compound and the PDE10 polypeptide; b) detecting binding of the compound to the PDE10 polypeptide; and c) identifying the compound as a specific binding partner of the PDE10 polypeptide. Preferably, methods of the invention identify specific binding partners that modulate activity of the PDE10 polypeptide. In one aspect, the methods identify compounds that inhibits activity of the PDE10 polypeptide. In another aspect, the methods identify compounds that enhance activity of the PDE10 polypeptide.
The invention also provides methods to identify a specific binding partner compound of the PDE10 polynucleotide of the invention comprising the steps of: a) contacting the PDE10 polynucleotide with a compound under conditions which permit binding between the compound and the PDE10 polynucleotide; b) detecting binding of the compound to the PDE10 polynucleotide; and c) identifying the compound as a specific binding partner of the PDE10 polynucleotide. Preferably, the methods identify specific binding partner compounds that modulate expression of a PDE10 polypeptide encoded by the PDE10 polynucleotide. In one aspect, method of the invention identify compounds that inhibit expression of the PDE10 polypeptide. In another aspect, methods of the invention identify compounds that enhance expression of the PDE10 polypeptide.
Binding partner compounds identified by methods of the invention are also contemplated, as are compositions comprising the compound. The invention further comprehends use of binding partner compounds of the invention in production of medicaments for the treatment of PDE10-related disorders.
The present invention provides polypeptides and underlying polynucleotides for a novel PDE family designated PDE10. The PDE10 family is distinguished from previously known PDE families in that it shows a lower degree of sequence homology than would be expected for a member of a known family of PDEs and it is not sensitive to inhibitors that are known to be specific for previously identified PDE families. Outside of the catalytic region of the protein, PDE10 shows little homology to other known PDEs. Even over the catalytic region, PDE10 amino acid sequence identity is less than 40% when compared with the same region in known PDEs. The invention includes both naturally occurring and non-naturally occurring PDE10 polynucleotides and polypeptide products thereof. Naturally occurring PDE10 products include distinct gene and polypeptide species within the PDE10 family; these species include those which are expressed within cells of the same animal as well as corresponding species homologs expressed in cells of other animals. Within each PDE10 species, the invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts. Non-naturally occurring PDE10 products include variants of the naturally occurring products such as analogs (i.e., wherein one or more amino acids are added, substituted, or deleted) and those PDE10 products which include covalent modifications (i.e., fusion proteins, glycosylation variants, and the like).
The present invention provides novel purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding human PDE10s. DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and includes allelic variants of the preferred polynucleotide of the invention. Genomic DNA of the invention is distinguishable from genomic DNAs encoding polypeptides other than PDE10 in that it includes the PDE10 coding region as defined by PDE10 cDNA of the invention. The invention therefore provides structural, physical, and functional characterization for genomic PDE10 DNA. Allelic variants are known in the art to be modified forms of a wild type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are inherently naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation). xe2x80x9cSynthesized,xe2x80x9d as used herein and is understood in the art, refers to purely chemical, as opposed to enzymatic, methods for producing polynucleotides. xe2x80x9cWhollyxe2x80x9d synthesized DNA sequences are therefore produced entirely by chemical means, and xe2x80x9cpartiallyxe2x80x9d synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. A preferred DNA sequence encoding a human PDE10 polypeptide is set out in SEQ ID NO: 1. The worker of skill in the art will readily appreciate that the preferred DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO: 1 along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson-Crick base paring rules for DNA. Also preferred are polynucleotides encoding the PDE10 polypeptide of SEQ ID NO: 2.
The disclosure of a full length polynucleotide encoding a PDE10 polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotide. The invention therefore provides fragments of PDE10-encoding polynucleotides of the invention comprising at least 10 to 20, and preferably at least 15, nucleotides, however, the invention comprehends fragments of various lengths. Preferably, fragment polynucleotides of the invention comprise sequences unique to the PDE10-encoding polynucleotide sequence, and therefore hybridize under stringent or preferably moderate conditions only (i.e., xe2x80x9cspecificallyxe2x80x9d) to polynucleotides encoding PDE10, or PDE10 polynucleotide fragments containing the unique sequence. Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs made available in public sequence databases.
The invention also provides fragment polynucleotides that are conserved in one or more polynucleotides encoding members of the PDE10 family of polypeptides. Such fragments include sequences characteristic of the family of PDE10 polynucleotides, and are also referred to as xe2x80x9csignature sequences.xe2x80x9d The conserved signature sequences are readily discernable following simple sequence comparison of polynucleotides encoding members of the PDE10 family. Fragments of the invention can be labeled in a manner that permits their detection, and radioactive and non-radioactive labeling are comprehended. Fragment polynucleotides are particularly useful as probes for detection of full length or other fragment PDE10 polynucleotides. One or more fragment polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding PDE10, or used to detect variations in a polynucleotide sequence encoding PDE10.
The invention further embraces species homologs, preferably mammalian, of the human PDE10 DNA. The polynucleotide sequence information provided by the invention makes possible the identification and isolation of polynucleotides encoding related mammalian PDE10 molecules by well known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to PDE10 and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of PDE10.
The invention also embraces DNA sequences encoding PDE10 species which hybridize under moderately stringent conditions to the non-coding strands, or complements, of the polynucleotide in any one of SEQ ID NOs: 1, 18, 20, and 22. DNA sequences encoding PDE10 polypeptides which would hybridize thereto but for the redundancy of the genetic code are contemplated by the invention. Exemplary moderate hybridization conditions are as follows: hybridization at 65xc2x0 C. in 3xc3x97SSC, 0.1% Sarkosyl, and 20 mM sodium phosphate, pH 6.8, and washing at 65xc2x0 C. in 2xc3x97SSC with 0.1% SDS. Exemplary high stringency conditions would include a final wash in 0.2xc3x97SSC/0.1% SDS, at 65xc2x0 C. to 75xc2x0 C. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausebel, et al. (Eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp.6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
Autonomously replicating recombinant expression constructions such as plasmid and viral DNA vectors incorporating PDE10 sequences are also provided. Expression constructs wherein PDE10-encoding polynucleotides are operatively-linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.
According to another aspect of the invention, host cells are provided, including procaryotic and eucaryotic cells, either stably or transiently transformed with DNA sequences of the invention in a manner which permits expression of PDE10 polypeptides of the invention. Expression systems of the invention include bacterial, yeast, fungal, viral, invertebrate, and mammalian cells systems. Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with PDE10. Host cells of the invention are also conspicuously useful in methods for large scale production of PDE10 polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by, for example, immunoaffinity purification.
Knowledge of PDE10 DNA sequences allows for modification of cells to permit, or increase, expression of endogenous PDE10. Cells can be modified (e.g., by homologous recombination) to provide increased PDE10 expression by replacing, in whole or in part, the naturally occurring PDE10 promoter with all or part of a heterologous promoter so that the cells express PDE10 at higher levels. The heterologous promoter is inserted in such a manner that it is operatively-linked to PDE10 encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. The invention also comprehends that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the PDE10 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the PDE10 coding sequences in the cells.
The DNA sequence information provided by the present invention also makes possible the development through, e.g. homologous recombination or xe2x80x9cknock-outxe2x80x9d strategies [Capecchi, Science 244:1288-1292 (1989)], of animals that fail to express functional PDE10 or that express a variant of PDE10. Such animals are useful as models for studying the in vivo activities of PDE10 and modulators of PDE10.
The invention also provides purified and isolated mammalian PDE10 polypeptides as set out in SEQ ID NOs: 2, 19, 21, and 23. Presently preferred is a PDE10 polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2. The invention embraces PDE10 polypeptides encoded by a DNA selected from the group consisting of: a) the DNA sequence set out in SEQ ID NOs:1, 18, 20, or 22; b) a DNA molecule which hybridizes under stringent conditions to the noncoding strand of the protein coding portion of (a); and c) a DNA molecule that would hybridize to the DNA of (a) but for the degeneracy of the genetic code. The invention also embraces polypeptide fragments of the sequences set out in SEQ ID NOs: 2, 19, 21, or 23 wherein the fragments maintain biological or immunological properties of a PDE10 polypeptide. Preferred polypeptide fragments display antigenic properties unique to or specific for the PDE10 family of polypeptides. Fragments of the invention can be prepared by any the methods well known and routinely practiced in the art, having the desired biological and immunological properties.
The invention embraces polypeptides have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% identity and/or homology to the preferred PDE10 polypeptide on the invention. Percent amino acid sequence xe2x80x9cidentityxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the PDE10 sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the PDE10 sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity. Conservative substitutions can be defined as set out below.
PDE10 polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of various host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. PDE10 products of the invention may be full length polypeptides, biologically or immunologically active fragments, or variants thereof which retain specific PDE10 biological or immunological activity. Variants may comprise PDE10 polypeptide analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added: (1) without loss of one or more of the biological activities or immunological characteristics specific for PDE10; or (2) with specific disablement of a particular biological activity of PDE10.
Variant products of the invention include mature PDE10 products, i.e., PDE10 products wherein leader or signal sequences are removed, and having additional, non-naturally occurring, amino terminal residues. PDE10 products having an additional methionine residue at position xe2x88x921 (Metxe2x88x921-PDE10) are contemplated, as are PDE10 products having additional methionine and lysine residues at positions xe2x88x922 and xe2x88x921 (Metxe2x88x922-Lysxe2x88x921-PDE10). Variants of these types are particularly useful for recombinant protein production in bacterial cell types.
The invention also embraces PDE10 variants having additional amino acid residues that result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide such as a glutathione-S-transferase (GST) fusion product provide the desired polypeptide having an additional glycine residue at position xe2x88x921 as a result of cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.
Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Conservative substitutions are recognized in the art to classify amino acids according to their related physical properties and can be defined as set out in Table I (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996).
Alternatively, conservative amino acids can be grouped as defined in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y. (1975), pp.71-77] as set out in Table II.
The invention further embraces PDE10 products modified to include one or more water soluble polymer attachments. Particularly preferred are PDE10 products covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the PDE10 products, or randomly attached to one or more side chains of the polypeptide.
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, human antibodies CDR-grafted antibodies, or otherwise xe2x80x9chumanizedxe2x80x9d antibodies, antigen binding antibody domains including Fab, Fabxe2x80x2, F(abxe2x80x2)2, Fv, or single variable domains, and the like) and other binding proteins specific for PDE10 products or fragments thereof. Specific binding proteins can be developed using isolated or recombinant PDE10 products, PDE10 variants, or cells expressing such products. The term xe2x80x9cspecific forxe2x80x9d indicates that the variable regions of the antibodies recognize and bind PDE10 polypeptides exclusively (i.e., able to distinguish PDE10 polypeptides from the superfamily of PDE polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (eds), Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the PDE10 polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, PDE10 polypeptides. As with antibodies that are specific for full length PDE10 polypeptides, antibodies of the invention that recognize PDE10 fragments are those which can distinguish PDE10 polypeptides from the superfamily of PDE polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.
Binding proteins are useful for purifying PDE10 products and detection or quantification of PDE10 products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of PDE10, especially those activities involved in signal transduction. Anti-idiotypic antibodies specific for anti-PDE10 antibodies are also contemplated.
The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for PDE10 makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding PDE10 and PDE10 expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under moderately to highly stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of PDE10; allelic variants are known in the art to include structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to PDE10. Similarly, non-human species genes encoding proteins homologous to PDE10 can also be identified by Southern and/or PCR analysis and useful in animal models for PDE10-related disorders. As an alternative, complementation studies can be useful for identifying other human PDE10 products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of PDE10. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express PDE10. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a PDE10 locus that underlies a disease state or states.
The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of PDE10s. DNA and amino acid sequence information for PDE10 also permits identification of binding partner compounds with which a PDE10 polypeptide or polynucleotide will interact. Binding partner compounds include proteins and non-protein compounds such as small molecules. Agents that modulate (i.e., increase, decrease, or block) PDE10 activity or expression may be identified by incubating a putative modulator with a PDE10 polypeptide or polynucleotide and determining the effect of the putative modulator on PDE10 phosphodiesterase activity or expression. The selectivity of a compound that modulates the activity of the PDE10 can be evaluated by comparing its binding activity on the PDE10 to its activity on other PDE enzymes. Cell based methods, such as di-hybrid assays to identify DNAs encoding binding compounds and split hybrid assays to identify inhibitors of PDE10 polypeptide interaction with a known binding polypeptide, as well as in vitro methods, including assays wherein a PDE10 polypeptide, PDE10 polynucleotide, or a binding partner are immobilized, and solution assays are contemplated under the invention.
Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to a PDE10 polypeptide or a PDE10-encoding nucleic acid, oligonucleotides which specifically bind to a PDE10 polypeptide or a PDE10 gene sequence, and other non-peptide compounds (e.g., isolated or synthetic organic and inorganic molecules) which specifically react with a PDE10 polypeptide or underlying nucleic acid. Mutant PDE10 polypeptides which affect the enzymatic activity or cellular localization of the wild-type PDE10 polypeptides are also contemplated by the invention. Presently preferred targets for the development of selective modulators include, for example: (1) regions of the PDE10 polypeptide which contact other proteins and/or localize the PDE10 polypeptide within a cell, (2) regions of the PDE10 polypeptide which bind substrate, (3) cyclic nucleotide-binding site(s) of the PDE10 polypeptide, (4) phosphorylation site(s) of the PDE10 polypeptide and (5) regions of the PDE10 polypeptide which are involved in multimerization of PDE10 subunits. Still other selective modulators include those that recognize specific PDE10 encoding and regulatory polynucleotide sequences. Modulators of PDE10 activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which PDE activity is known to be involved.
PDE10 polypeptides of the invention are particularly amenable to use in high throughput screening assays to identify binding partners, and preferably modulators. Cell based assays are contemplated, including yeast based assay systems as well as mammalian cell expression systems as described in Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634 (1997). Alternatively, automated and minaturized high throughput screening (HTS) assays, such as high density free format high density screening, as described in Houston and Banks, Curr. Opin. Biotehcnol. 8:734-740 (1997). Combinatorial libraries are particularly useful in high throughput screening assays.
There are a number of different libraries used for the identification of small molecule modulators, including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
Chemical libraries consist of structural analogs of known compounds or compounds that are identified as xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d via natural product screening. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opion. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate xe2x80x9chitxe2x80x9d (or xe2x80x9cleadxe2x80x9d) to optimize the capacity of the xe2x80x9chitxe2x80x9d to modulate activity.
Also made available by the invention are anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding PDE10. Full length and fragment anti-sense polynucleotides are provided. The worker of ordinary skill will appreciate that fragment anti-sense molecules of the invention include (i) those which specifically recognize and hybridize to PDE10 RNA (as determined by sequence comparison of DNA encoding PDE10 to DNA encoding other known molecules) as well as (ii) those which recognize and hybridize to RNA encoding variants in the PDE10 family of proteins. Antisense polynucleotides that hybridize to RNA encoding other members of the PDE10 family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of PDE10 by those cells expressing PDE10 mRNA.
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to PDE10 expression control sequences or PDE10 RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the PDE10 target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use according to the invention. The antisense oligonucleotides maybe further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at the 5xe2x80x2 end.
The invention further comprehends methods to modulate PDE10 expression through use of ribozymes. For a review, see Gibson and Shillitoe, Mol. Biotech. 7:125-137 (1997). Ribozyme technology can be utilized to inhibit translation of PDE10 mRNA in a sequence specific manner through (i) the hybridization of a complementary RNA to a target mRNA and (ii) cleavage of the hybridized mRNA through nuclease activity inherent to the complementary strand. Ribozymes can identified by empirical methods but more preferably are specifically designed based on accessible sites on the target mRNA [Bramlage, et al., Trends in Biotech 16:434-438 (1998).] Delivery of ribozymes to target cells can be accomplished using either exogenous or endogenous delivery techniques well known and routinely practiced in the art. Exogenous delivery methods can include use of targeting liposomes or direct local injection. Endogenous methods include use of viral vectors and non-viral plasmids.
Ribozymes can specifically modulate expression of PDE10 when designed to be complementary to regions unique to a polynucleotide encoding PDE10. xe2x80x9cSpecifically modulatexe2x80x9d therefore is intended to mean that ribozymes of the invention recognizes only a polynucleotide encoding PDE10. Similarly, ribozymes can be designed to modulate expression of all or some of the PDE10 family of proteins. Ribozymes of this type are designed to recognize polynucleotide sequences conserved in all or some of the polynucleotides which encode the family of proteins.
The invention further embraces methods to modulate transcription of PDE10 through use of oligonucleotide-directed triplet helix formation. For a review, see Lavrosky, et al., Biochem. Mol. Med. 62:11-22 (1997). Triplet helix formation is accomplished using sequence specific oligonucleotides which hybridize to double stranded DNA in the major groove as defined in the Watson-Crick model. Hybridization of a sequence specific oligonucleotide can thereafter modulate activity of DNA-binding proteins, including, for example, transcription factors and polymerases. Preferred target sequences for hybridization include promoter and enhancer regions to permit transcriptional regulation of PDE10 expression. Oligonucleotides which are capable of triplet helix formation are also useful for site-specific covalent modification of target DNA sequences. Oligonucleotides useful for covalent modification are coupled to various DNA damaging agents as described in Lavrovsky, et al. [supra].
The invention comprehends mutations in the PDE10 gene that result in loss of normal function of the PDE10 gene product and underlie human disease states in which failure of the PDE10 is involved. Gene therapy to restore PDE10 activity would thus be indicated in treating those disease states. Delivery of a functional PDE10 gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357:455-460 (1992). Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of PDE10 will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of PDE10.
Identification of modulators of PDE10 expression and/or biological activity provides methods to treat disease states that arise from aberrant PDE10 activity. Modulators may be prepared in compositions for administration, and preferably include one or more pharmaceutically acceptable carriers, such as pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma. The modulator compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the composition into the recipient organism and, particularly, when the composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets. The compositions may be introduced into the subject by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, or subcutaneous injection; by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time.
The invention also embraces use of a PDE10 polypeptide, a PDE10 polynucleotide, or a binding partner thereof in production of a medicament for treatment of a PDE10-related biological disorder.
The present invention is illustrated by the following examples relating to the isolation of a polynucleotide encoding a PDE10 polypeptide and expression thereof. Example 1 describes identification of an EST encoding a partial PDE10 polypeptide and isolation of a full length PDE10-encoding clone. Example 2 relates to Northern blot analysis of PDE10 expression. Example 3 addresses chromosome mapping of PDE10. Example 4 describes expression and characterization of a recombinant PDE10 polypeptide. Example 5 describes production of anti-PDE10 antibodies. Example 6 provides an analysis of PDE10 expression using in situ hybridization. Example 7 relates to high throughput screening to identify inhibitors of PDE10.